The release of excitatory amino acids (EAAs) -- glutamate, and aspartate - - during anoxic and ischemic events in the central nervous system (such as stroke) contributed to the subsequent neuronal injury. Recent therapeutic strategies that focus on blockade of postsynaptic EAA receptors have proven to be problematic because of the toxicity of these agents. To develop alternative therapeutic approaches, the mechanisms whereby excitotoxic glutamate is released during such hypometabolic states will be studied. In hippocampal slices, hypoxia causes an increase mEPSC frequency and resting glutamatergic tone (two measures of the amount of potentially excitotoxic glutamate in the tissue) in CA1 neurons of the hippocampal slices in vitro by whole-cell patch clamp. The general hypothesis of this proposal is that the vesicular pools of glutamate which are released during hypoxia are a result of a complex series of events starting with an increase in NMDA receptor stimulation resulting from decreased glutamate uptake. This results in elevations in postsynaptic calcium leading to the production of NO or CO. These gaseous compounds act as local retrograde messengers promoting further release of glutamate by the presynaptic mobilization of intracellular stores of calcium. This leads to further NMDA activation, closing a positive feedback loop resulting in a subsequent massive vesicular glutamate release. Aim 1 will examine the relation between an increase in vesicular glutamate release and tonic glutamate tone and will test the hypothesis that it is related to NMDA receptor activation. Aim 2 will examine the hypothesis that the elevation in extracellular glutamate results from intracellular calcium mobilization. Aim 3 will examine the hypothesis that the release of glutamate results from an increase in gaseous messengers. Aim 4 will examine the causal relationship between the individual elements of the general hypothesis as stated above.